Western Blot Analysis of Enamel Proteins

The first lower molars dissected from 4-day-old WT, ENAM^Rgsc514 heterozygous, and ENAM^Rgsc514 homozygous mice were ground into powder in liquid nitrogen and lysed using RIPA buffer (ThermoFisher Scientific, Waltham, MA, USA) containing a proteinase inhibitor cocktail (Roche, Indianapolis, IN, USA). After quantitation by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific), the lysates containing equal amounts of total proteins from each group were loaded on sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) and analyzed by Western immunoblotting using the antibodies anti-ENAMN-terminus (1:1 600),¹³ anti-AMBN C-terminus (1:2 000, SC-50534, Santa Cruz Biotechnology), anti-AMEL (1:2 000, SC-32892, Santa Cruz Biotechnology), and β-ACTIN(1:3 000, SC-47778, Santa Cruz Biotechnology), using the methods described previously.^{6 (link), 14 (link), 15 (link), 16 (link)}